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Electrophoretic staining of histones in polyacrylamide gels
\x
4l,YTI(‘AI,
HIO(‘tIEMISTKY
Electrophoretic
55,
Staining
620-622
(1973)
of Histones
in Polyacrylamide
Gels
Analytical polyacrylamide gel electrophoresis is a useful tool for testing the purity of historic fractions during isolation. Several mcthotls for the electrophorcsis of histones in polyacrylamiclc gel (PAGI are found in the literature (l-3). In a recent publication, Ahlroth and Mutt (4) describe a method for the visualization of zones in PACT by electrophoretic migration of the dye (amid0 black) counter to the direction of migration of basic polypeptides from the int,estinal wall. ilmido black is an acid azo clyc containing two sulfonic group. In wit1 media the dye anion is clcctrostntically attnchcd to basic groulw of pot&is. The present paper deals with this rapid stainin g tcchniquc applied to PAG elcctrophoresis of histone, 9 in various conditions. The method was found to be precise and well reproducible and to yield banding pattcms comparable to those obtained with conventional staining procedurw. Methods. Histone fractions were prepared from rat liver (RIcthod 1 ) and calf thymus (Method 2) according to Johns (5). Whole historic from rat liver was pwpnrcd according t’o Piha et al. (6). PA($ clertrophoresis was performed with Shandon SEA 2734 and Cannlco 1\1 100 apparatusrr. Standard tube sizes 5 X 75 mm wcw used, except whore otherwise statctl. The chemicals were obtained from Easknan Kodak Company. PAG electrophoresis at pH 2.4 according to Johns (1). Twnty-pcrccnt gels were prerun for 2 hr at 2 mA per gel before application of the sample. Semi-mirrotubes of 2 X 75 mm were prcrun at 0.75 mA Iwr gel. After application of the sample (5-1.5 pg.), thr gcle wert run for 45 min (s-m tubes, sarnplr 2-5 ;I,%,for 20 min) in 0.01 M acct#ic acid. One millitcr of 1% amid0 black 10 13 was added per 100 ml of rathotlc solution and the dye was run for 15-20 min at the same currtwt. The cathode solution was then changed for fresh 0.01 M acetic acid, and the> clcctrophoreeis wan rontin\lcti until thcx CXCCRS tlyc had rcachcd the anode compartment. PAG
electrophoresis
nt pH 2.7 according
to Pnnyim
and
Chnlkley
(21.
Elcctrophoresis was performed in 15% gvls containing 2.5 $1 urea, pH 2.7. The gels were prcrun for 2 hr or to constant voltage in 0.9 M acetic acid. Longer tubes, 5 X 200 mm, were occasionally used. Thr samples were run for l-2 hr (the longer h~hcs for 10-20 hr‘l before the dye was 620
Copyright, .4lI rights
@ 1973 hy Academic Press, Inc. of reprodurtion in nny form wserwd.
added to the cathode buffer. Staining and destaining were performed as described above. Disc electrophoresis according to Lindh rind Brccntmrk (3). Fiftccn0.1 >I percent polyacrylaiuicle gels n-it11 pI-I of 5.1 ; anode buffer: cntliotle I)uffclr: 0.06 ~1 I<-acchtc, /3-alanine, 03 X 10-” >I I<-acetatc; l)H 5.1. In this modification thr amiclo black 1OB (O.Ol(;/ J W:~Y xtltled to the cathode huft’er at the onset of the run. Tllca cat8hotlc solution was chnngctl for frwh catliotlr~ hufcr :ui(l dcst:Gniiig m-as pcrformccl nhcn tjlit, clyc reached tlic lnwteins, n-liicli war wc’ii :LS the form:~tioii of :I sli:Lq) elk Imirl at tllc point of contact. l~‘igurc 1 rliows tlica Ix~utlirig ptteriis tniriecl from wrious liistoiiv hctions and ~vliol~~ histow prqx~rntions t)y c,l~,~troI)llorc~tic staiiiiiig ill ~~oly:wryl:miidc gala. The zone formctl n-err very slxq~ anal distiu(~t, ~~11~1 thus tllcl rcsolutiou of th variow compoucutr in n-liol~t histone prcqwxtiow war not mcak-
622
SHORT
COMMUNICATIONS
ened although the fastest moving zones became fixed at a time when the slower ones were still moving. By using internal markers, such as serum albumin, we could easily compare the relative mobilities of t,he various histone fractions. ,4n obvious advantage of this method is the ease and relative speed of staining and destaining. Yet another modification of this staining principle has been reported by Lushnikov and Balandin (7). They describe a method for countercurrent coloring of histone fraction s in PAG electrophoresis at pH 4.0. Zones were visualized by reversing the tubes and running the dye from the end where the sample was originally applied. ACKSOWLEDGMENTS We thank Professor R. S. Piha our work, and Miss Tuula Ku&la
for his kind encouragenlent for her collaboraiion.
during
the
course
REFERENCES 1. JOHNS, E. W. (1967) Biochem. J. 104, 78. 2. PZ~NYIM, S.. AXD C~~LKLEI-, R. (1969) Arch. Hiochem. Biophys. 130, 337. 3. LINDH, N. O., AND BHANTMARIC. B. I,. 11966) in Methods in Biochelnical Anal>& (D. Glick, ed.), Vol. 14, p. 79. John Wiley & Sons Ltd., New York. 4. AHLROTH, A., .4x1 MUTT, V. (1969) A~nl. Riochem. 37, 125. 5. JOHNS, E. W. (1964) Biochem. J. 92, 55. 6. PIHA, R. S.. CU~~NOD, M., ANI) W.~IZLX:H, H. (1966) 1. Biol. Chem. 241, 2397. 7. LUSHNIKOV, A. A., .wn B.+L.~NIux. I. G. (1971) Lab. Delo. 9, 522. HELENA PIRK~O Department of Biochemistry University of Oulu Oulu. Finland Received Jnnuary 2.7, 19YS;
accepted
May
21, 1.77~
HOLMBERG HUTTUNI-X
of
4l,YTI(‘AI,
HIO(‘tIEMISTKY
Electrophoretic
55,
Staining
620-622
(1973)
of Histones
in Polyacrylamide
Gels
Analytical polyacrylamide gel electrophoresis is a useful tool for testing the purity of historic fractions during isolation. Several mcthotls for the electrophorcsis of histones in polyacrylamiclc gel (PAGI are found in the literature (l-3). In a recent publication, Ahlroth and Mutt (4) describe a method for the visualization of zones in PACT by electrophoretic migration of the dye (amid0 black) counter to the direction of migration of basic polypeptides from the int,estinal wall. ilmido black is an acid azo clyc containing two sulfonic group. In wit1 media the dye anion is clcctrostntically attnchcd to basic groulw of pot&is. The present paper deals with this rapid stainin g tcchniquc applied to PAG elcctrophoresis of histone, 9 in various conditions. The method was found to be precise and well reproducible and to yield banding pattcms comparable to those obtained with conventional staining procedurw. Methods. Histone fractions were prepared from rat liver (RIcthod 1 ) and calf thymus (Method 2) according to Johns (5). Whole historic from rat liver was pwpnrcd according t’o Piha et al. (6). PA($ clertrophoresis was performed with Shandon SEA 2734 and Cannlco 1\1 100 apparatusrr. Standard tube sizes 5 X 75 mm wcw used, except whore otherwise statctl. The chemicals were obtained from Easknan Kodak Company. PAG electrophoresis at pH 2.4 according to Johns (1). Twnty-pcrccnt gels were prerun for 2 hr at 2 mA per gel before application of the sample. Semi-mirrotubes of 2 X 75 mm were prcrun at 0.75 mA Iwr gel. After application of the sample (5-1.5 pg.), thr gcle wert run for 45 min (s-m tubes, sarnplr 2-5 ;I,%,for 20 min) in 0.01 M acct#ic acid. One millitcr of 1% amid0 black 10 13 was added per 100 ml of rathotlc solution and the dye was run for 15-20 min at the same currtwt. The cathode solution was then changed for fresh 0.01 M acetic acid, and the> clcctrophoreeis wan rontin\lcti until thcx CXCCRS tlyc had rcachcd the anode compartment. PAG
electrophoresis
nt pH 2.7 according
to Pnnyim
and
Chnlkley
(21.
Elcctrophoresis was performed in 15% gvls containing 2.5 $1 urea, pH 2.7. The gels were prcrun for 2 hr or to constant voltage in 0.9 M acetic acid. Longer tubes, 5 X 200 mm, were occasionally used. Thr samples were run for l-2 hr (the longer h~hcs for 10-20 hr‘l before the dye was 620
Copyright, .4lI rights
@ 1973 hy Academic Press, Inc. of reprodurtion in nny form wserwd.
added to the cathode buffer. Staining and destaining were performed as described above. Disc electrophoresis according to Lindh rind Brccntmrk (3). Fiftccn0.1 >I percent polyacrylaiuicle gels n-it11 pI-I of 5.1 ; anode buffer: cntliotle I)uffclr: 0.06 ~1 I<-acchtc, /3-alanine, 03 X 10-” >I I<-acetatc; l)H 5.1. In this modification thr amiclo black 1OB (O.Ol(;/ J W:~Y xtltled to the cathode huft’er at the onset of the run. Tllca cat8hotlc solution was chnngctl for frwh catliotlr~ hufcr :ui(l dcst:Gniiig m-as pcrformccl nhcn tjlit, clyc reached tlic lnwteins, n-liicli war wc’ii :LS the form:~tioii of :I sli:Lq) elk Imirl at tllc point of contact. l~‘igurc 1 rliows tlica Ix~utlirig ptteriis tniriecl from wrious liistoiiv hctions and ~vliol~~ histow prqx~rntions t)y c,l~,~troI)llorc~tic staiiiiiig ill ~~oly:wryl:miidc gala. The zone formctl n-err very slxq~ anal distiu(~t, ~~11~1 thus tllcl rcsolutiou of th variow compoucutr in n-liol~t histone prcqwxtiow war not mcak-
622
SHORT
COMMUNICATIONS
ened although the fastest moving zones became fixed at a time when the slower ones were still moving. By using internal markers, such as serum albumin, we could easily compare the relative mobilities of t,he various histone fractions. ,4n obvious advantage of this method is the ease and relative speed of staining and destaining. Yet another modification of this staining principle has been reported by Lushnikov and Balandin (7). They describe a method for countercurrent coloring of histone fraction s in PAG electrophoresis at pH 4.0. Zones were visualized by reversing the tubes and running the dye from the end where the sample was originally applied. ACKSOWLEDGMENTS We thank Professor R. S. Piha our work, and Miss Tuula Ku&la
for his kind encouragenlent for her collaboraiion.
during
the
course
REFERENCES 1. JOHNS, E. W. (1967) Biochem. J. 104, 78. 2. PZ~NYIM, S.. AXD C~~LKLEI-, R. (1969) Arch. Hiochem. Biophys. 130, 337. 3. LINDH, N. O., AND BHANTMARIC. B. I,. 11966) in Methods in Biochelnical Anal>& (D. Glick, ed.), Vol. 14, p. 79. John Wiley & Sons Ltd., New York. 4. AHLROTH, A., .4x1 MUTT, V. (1969) A~nl. Riochem. 37, 125. 5. JOHNS, E. W. (1964) Biochem. J. 92, 55. 6. PIHA, R. S.. CU~~NOD, M., ANI) W.~IZLX:H, H. (1966) 1. Biol. Chem. 241, 2397. 7. LUSHNIKOV, A. A., .wn B.+L.~NIux. I. G. (1971) Lab. Delo. 9, 522. HELENA PIRK~O Department of Biochemistry University of Oulu Oulu. Finland Received Jnnuary 2.7, 19YS;
accepted
May
21, 1.77~
HOLMBERG HUTTUNI-X
of